Typing system containing 29 groups of X chromosome STR gene loci and primer composition and application thereof

Abstract

The invention relates to the technical field of forensic medicine, and particularly discloses a typing system containing 29 groups of X chromosome STR gene loci as well as a primer composition and application of the typing system. Through screening and evaluation, a typing system containing 29 groups of X chromosome STR gene loci is provided, and the typing system comprises 19 independent X-STRs and 47 X-STRs including 10 X-STR linkage clusters. The typing system provided by the invention can be used for further identification when the relationship between two individuals is determined to belong to a secondary genetic relationship and various genetic relationships cannot be identified by autosomal loci. Compared with the typing system of the X chromosome STR gene locus in the prior art, the typing system provided by the invention has higher accuracy, is suitable for more complicated genetic relationship identification situations, and provides more reliable evidence support for genetic relationship identification.

Description

Technical Field

[0001] This invention relates to the field of forensic medicine technology, and specifically discloses a typing system containing 29 X chromosome STR loci, its primer composition, and its applications. Background Technology

[0002] STR (Short Tandem Repeat) gene loci consist of short tandem repeat sequences of 3–7 base pairs in length. These repeat sequences are widely distributed in the human genome and serve as highly polymorphic markers, often referred to as the DNA fingerprint of a cell. STRs have been widely used in kinship testing, individual identification, forensic identification, and traffic accident investigation.

[0003] Although the practicality of autosomal STRs (A-STRs) and Y-chromosome STRs (Y-STRs) has been well demonstrated, the research and practical application of X-chromosome STRs (X-STRs) have progressed relatively slowly. Most of the STRs disclosed in existing technologies are autosomal STRs. While they can effectively solve common problems in paternity testing and full sibling identification, autosomal STRs are no longer sufficient for identifying half-siblings, grandparents and grandchildren, uncles and nephews, or aunts and nephews. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a typing system comprising 29 X-chromosome STR loci, its primer composition, and its applications. This typing system can assist autosomal loci in complex kinship identification, especially in the fine differentiation of second-degree relationships, exhibiting higher accuracy and playing a unique role that autosomal loci cannot easily replace.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a typing system comprising 29 sets of X chromosome STR loci, wherein the 29 sets of X chromosome STR loci include 19 independent X-STRs and 10 X-STR linkage clusters;

[0007] Among them, 19 independent X-STRs include DXS9902, DXS6795, DXS9907, DXS6810, GATA144D04, DXS981, DXS6800, DXS6803, DXS9898, DXS9905, DXS6801, DXS6799, DXS7133, DXS6804, GATA172D05, DXS7130, GATA165B12, GATA31E08 and DXS7127;

[0008] The 10 X-STR linkage clusters include 10 linkage clusters, from linkage cluster 1 to linkage cluster 10.

[0009] Cluster 1 includes DXS680703, DXS6807, DXS680706, and DXS680710; Cluster 2 includes DXS9895 and DXS9906; Cluster 3 includes DXS10135 and DXS8378; Cluster 4 includes DXS10076 and DXS10078; Cluster 5 includes DXS10161, DXS10160, and DXS10159; Cluster 6... The chain includes DXS7132, DXS10079, DXS10074 and DXS10075, chain 7 includes DXS6809 and DXS6789, chain 8 includes DXS7424 and DXS101, chain 9 includes DXS10103, HPRTB and DXS10101, and chain 10 includes DXS8377, DXS10146, DXS10147 and DXS7423.

[0010] This invention provides a typing system comprising 29 X-chromosome STR loci (X-STRs) through screening and evaluation. This system includes 47 X-STRs, comprising 19 independent X-STRs and 10 linked X-STR clusters. The typing system provided by this invention can further identify relationships when two individuals are determined to be second-degree related, but autosomal loci cannot distinguish between multiple kinship relationships.

[0011] Compared with the existing X chromosome STR locus typing system, the typing system provided by this invention has higher accuracy, is applicable to more complex kinship identification situations, and provides more reliable evidence for kinship identification.

[0012] Preferably, the typing system further includes five X chromosome STR loci, specifically DXS10162, DXS10163, DXS680705, DXS680708 and DXS680709.

[0013] The preferred typing system contains 52 X-chromosome STR loci, specifically DXS10074, DXS10075, DXS10076, DXS10078, DXS10079, DXS101, DXS10101, DXS10103, DXS10135, DXS10146, DXS10147, DXS10159, DXS10160, DXS10161, DXS10162, DXS10163, DXS6789, DXS6795, DXS6799, DXS6800, DXS6801, DXS6803, DXS6804, DXS6807, and DXS6808. 0703, DXS680705, DXS680706, DXS680708, DXS680709, DXS680710, DXS6809, DXS6810, DXS7127, DXS7130, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS981, DXS9895, DXS9898, DXS9902, DXS9905, DXS9906, DXS9907, GATA144D04, GATA165B12, GATA172D05, GATA31E08 and HPRTB.

[0014] The specific information of the 52 X chromosome STR gene loci is shown in Table 1.

[0015] Table 1

[0016]

[0017]

[0018] In a second aspect, the present invention provides a primer composition for the typing system comprising 29 X chromosome STR loci as described in the first aspect, wherein the primer composition comprises primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.94.

[0019] Preferably, the primer composition further includes primers with nucleotide sequences as shown in SEQ ID NO.95 to SEQ ID NO.104.

[0020] The sequences of the primer compositions provided by this invention are shown in Table 2.

[0021] Table 2

[0022]

[0023]

[0024]

[0025] Note: In primer sequences, "F" indicates "forward" and "R" indicates "reverse".

[0026] Thirdly, the present invention provides a kit comprising a primer composition for amplifying the genotyping system comprising 29 X chromosome STR loci as described in the first aspect.

[0027] Preferably, the primer composition comprises the primers shown in SEQ ID NO.1 to SEQ ID NO.94 or the primers shown in SEQ ID NO.1 to SEQ ID NO.104.

[0028] For example, in addition to the primer composition described above, the kit also includes an IGT-EM808 polymerase mixture, enhancement buffer NB, enhancement buffer M, YF buffer B, an IGT-I5 tag, and an IGT-I7 tag.

[0029] The kit provided by this invention is suitable for next-generation sequencing. The kit has advantages such as high genotyping accuracy, good reproducibility, high sensitivity, strong species specificity, excellent inhibitor tolerance and anti-degradation ability.

[0030] Fourthly, the present invention provides the application of the typing system comprising 29 X chromosome STR loci described in the first aspect in non-diagnostic individual identification or kinship identification.

[0031] The typing system provided by this invention can meet the requirements of individual identification and kinship determination, and provides a theoretical and experimental research basis for using X chromosome STR loci to solve individual identification or complex kinship determination.

[0032] Preferably, the kinship relationship is a second-degree kinship relationship.

[0033] The second-degree kinship relationships include female-female kinship, male-female kinship, and male-male kinship.

[0034] The female-female kinship relationships include the following three groups: Group 1: grandmother-granddaughter, half-sisters; Group 2: aunt-niece; Group 3: maternal grandmother-granddaughter, half-sisters, aunt-niece.

[0035] The male-female kinship relationships include the following four groups: Group 4: Aunt-nephew; Group 5: Half-siblings, maternal grandmother-grandson, maternal grandfather-granddaughter, uncle-niece; Group 6: Uncle-niece; Group 7: Grandmother-grandson, grandfather-granddaughter, aunt-nephew, half-siblings.

[0036] The male-male kinship relationships include the following three groups: Group 8: half-brothers (same mother, different fathers), maternal grandfather-grandson; Group 9: maternal uncle-nephew; Group 10: paternal grandfather-grandson, uncle-nephew, half-brothers (same father, different mothers).

[0037] After determining that two individuals are in a secondary relationship, it is difficult to determine the degree of kinship using autosomal loci. However, the typing system provided by this invention, which includes 29 groups of X chromosome STR loci, can further differentiate between groups of different secondary kinship relationships.

[0038] For example, in second-degree related individuals in Group 10, the two individuals exhibit the same X chromosome behavior as unrelated individuals, but this invention can distinguish Group 10 from Group 8 / 9.

[0039] Fifthly, the present invention provides a method for identifying kinship for non-diagnostic purposes, the method comprising the following steps:

[0040] DNA was extracted from two samples to be tested, and the genotyping system containing 29 X chromosome STR loci as described in the first aspect was amplified. After sequencing by a next-generation sequencing platform, the data was analyzed to obtain the X chromosome STR locus allele genotypes of the samples and calculate the likelihood rate. The kinship was identified based on the likelihood rate results.

[0041] Preferably, the process of extracting DNA from two test samples, amplifying the genotyping system containing 29 X chromosome STR loci as described in the first aspect, and analyzing the data and calculating the likelihood after sequencing on a next-generation sequencing platform specifically includes: extracting and quantifying DNA from the peripheral blood of the two test samples; using the kit described in the third aspect, performing a first round of multiplex PCR amplification and magnetic bead purification, a second round of adapter sequence PCR reaction and magnetic bead purification of the product, followed by quantification and quality testing of the library, sequencing on a next-generation sequencing platform, analyzing and obtaining the X chromosome STR locus allele genotyping of the samples, and calculating the likelihood based on the population frequency of the corresponding alleles.

[0042] The genotyping system provided by this invention, which includes 29 X chromosome STR loci, provides accurate genotyping, enables further mining of X chromosome genetic information, and improves the genetic polymorphism of the genotyping system.

[0043] The kit developed based on the above analysis system is suitable for next-generation sequencing and has advantages such as high genotyping accuracy, good reproducibility, high sensitivity, strong species specificity, excellent inhibitor tolerance and anti-degradation ability.

[0044] The typing system significantly improves the accuracy of non-diagnostic kinship identification methods, solving the technical challenge in forensic identification where chromosomes often determine second-level kinship but fail to provide further precise differentiation. This invention offers higher identification efficiency for forensic X-STR applications in solving complex kinship identification and individual recognition. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is the library quality control image in effect 1 of the present invention;

[0047] Figure 2 This is a comparison chart of the number of alleles obtained based on gene length and gene sequence polymorphism in Example 2 of the present invention;

[0048] Figure 3 Example 3 of the present invention uses the confusion matrix diagram of the typing system containing 29 X chromosome STR loci provided in Example 1 to finely distinguish 22 kinds of secondary kinship;

[0049] Figure 4 Example 3 of the present invention uses the confusion matrix diagram provided in Example 2, which includes a typing system containing 29 X chromosome STR loci, to finely distinguish 22 kinds of secondary kinship.

[0050] Figure 5 Example 3 of the present invention uses the confusion matrix diagram of the typing system containing 32 X-STRs provided in Comparative Example 1 to finely distinguish 22 kinds of secondary kinship;

[0051] Figure 6 This is a second-order kinship pedigree chart drawn in Typical Case 2 of the present invention. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0053] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0054] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0055] Example 1

[0056] This invention provides a typing system comprising 29 sets of X-chromosome STR loci, wherein the 29 sets of X-chromosome STR loci include 47 X-STRs, including 19 independent X-STRs and 10 X-STR linkage clusters, as detailed below:

[0057] Among them, 19 independent X-STRs include DXS9902, DXS6795, DXS9907, DXS6810, GATA144D04, DXS981, DXS6800, DXS6803, DXS9898, DXS9905, DXS6801, DXS6799, DXS7133, DXS6804, GATA172D05, DXS7130, GATA165B12, GATA31E08 and DXS7127;

[0058] The 10 X-STR linkage clusters include 10 linkage clusters, from linkage cluster 1 to linkage cluster 10.

[0059] Cluster 1 includes DXS680703, DXS6807, DXS680706, and DXS680710; Cluster 2 includes DXS9895 and DXS9906; Cluster 3 includes DXS10135 and DXS8378; Cluster 4 includes DXS10076 and DXS10078; Cluster 5 includes DXS10161, DXS10160, and DXS10159; Cluster 6... The chain includes DXS7132, DXS10079, DXS10074 and DXS10075, chain 7 includes DXS6809 and DXS6789, chain 8 includes DXS7424 and DXS101, chain 9 includes DXS10103, HPRTB and DXS10101, and chain 10 includes DXS8377, DXS10146, DXS10147 and DXS7423.

[0060] Example 2

[0061] This invention provides a genotyping system containing 29 X-chromosome STR loci. Based on the genotyping system provided in Example 1, it further includes DXS10162, DXS10163, DXS680705, DXS680708, and DXS680709. The genotyping system provided in this embodiment contains 52 X-STRs, specifically DXS10074, DXS10075, DXS10076, DXS10078, DXS10079, DXS101, DXS10101, DXS10103, DXS10135, DXS10146, DXS10147, DXS10159, DXS10160, DXS10161, DXS10162, DXS10163, DXS6789, DXS6795, and DXS6... 799, DXS6800, DXS6801, DXS6803, DXS6804, DXS6807, DXS680703, DXS680705, DXS68070 6. DXS680708, DXS680709, DXS680710, DXS6809, DXS6810, DXS7127, DXS7130, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS981, DXS9895, DXS9898, DXS9902, DXS9905, DXS9906, DXS9907, GATA144D04, GATA165B12, GATA172D05, GATA31E08 and HPRTB.

[0062] The specific information of the 52 X chromosome STR gene loci is shown in Table 1 above.

[0063] Example 3

[0064] This invention provides a kit containing primer compositions for amplifying 47 X-STRs from Example 1 with paired-end specific primers. The primer compositions include primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.94, and information on different primers is shown in Table 2.

[0065] The kit also includes IGT-EM808 polymerase mixture, enhancement buffer NB, enhancement buffer M, YF buffer B, IGT-I5 tag, and IGT-I7 tag.

[0066] Example 4

[0067] This invention provides a kit containing primer compositions for amplifying 52 X-STRs with paired-end specific primers as shown in Example 2. The primer compositions include primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.104, and information on different primers is shown in Table 2.

[0068] The kit also includes IGT-EM808 polymerase mixture, enhancement buffer NB, enhancement buffer M, YF buffer B, IGT-I5 tag, and IGT-I7 tag.

[0069] Example 5

[0070] This invention provides a method for identifying kinship for non-diagnostic purposes, the method comprising the following steps:

[0071] DNA was extracted from peripheral blood of two test samples. The genetic information of the corresponding loci was amplified using the kits provided in Example 3 or 4. After sequencing by a next-generation sequencing platform, the data was analyzed to obtain the allele frequency data of the X chromosome STR loci of the samples and calculate the likelihood rate. The kinship was identified based on the likelihood rate results.

[0072] Comparative Example 1

[0073] This invention provides a comparative example of a typing system comprising 32 X-STRs, wherein the 32 X-STRs include DXS8378, DXS7423, DXS10148, DXS10159, DXS10134, DXS7424, DXS9902, DXS10162, DXS7132, DXS10079, DXS6789, GATA165B12, DXS101, and DXS10 075, DXS10103, DXS10101, HPRTB, DXS6809, GATA31E08, GATA172D05, DXS6803, DXS10074, DXS10135, DXS6810, DXS6795, DXS6800, DXS7133, DXS10164, DXS6807, DXS9895, DXS8377 and DXS981.

[0074] Example of effect 1

[0075] To verify whether the detection results of the kit are reliable, stable, and meet application requirements, this effect example evaluates the sequencing performance of the kit provided in Example 4, including the following two parts: first, the method of using the kit is provided; second, the sensitivity, repeatability, and specificity of the obtained results are evaluated, as follows:

[0076] I. How to use the reagent kit provided in Example 4

[0077] 1. Material preparation

[0078] Peripheral blood was collected from 128 unrelated Han Chinese individuals from Hebei Province (69 females and 59 males), all of whom signed informed consent forms. The positive control was DNA standard NA18530, and the negative control was nuclease-free water.

[0079] 2. DNA extraction and quantification

[0080] DNA was extracted using the QIAamp DNA Investigator Kit (QIAGEN, Germany) according to the kit's instructions.

[0081] The concentration of genomic DNA was quantified using the Qubit™ dsDNA HS Assay Kit (Introvigen, USA), and the preparation system is shown in Table 3 below.

[0082] Table 3

[0083] reagents Volume (μL) Qubit™ dsDNA HS Reagent 1 Qubit™ dsDNA HS Buffer 199

[0084] Add 190 μL of the mixture and 10 μL of standard to the standard tube; add 198 μL of the mixture and 2 μL of the sample DNA to be tested to the sample test tube; vortex to mix, incubate at room temperature for 2 minutes, and use Qubit... TM Quantitative analysis was performed using a 3.0 fluorescence quantitative analyzer.

[0085] 3. PCR amplification

[0086] Using extracted genomic DNA as a template, PCR amplification was performed, including the following steps:

[0087] 3.1 First round of multiplex PCR reaction:

[0088] (1) The PCR amplification system includes the following reagents and quantities, as shown in Table 4 below.

[0089] Table 4

[0090] PCR system Volume (μL) <![CDATA[ddH2O]]> 9-x Enhancer buffer NB(1N) 3.5 Enhancer buffer M 2.5 Primer pool 5 Genomic DNA (10 ng) x IGT-EM808 polymerase mixture 10 Total volume 30

[0091] (2) Vortex the PCR tubes to mix them, centrifuge briefly, and then place them in a thermal cycler. Run the PCR program, where the PCR program cycle parameters are shown in Table 5.

[0092] Table 5

[0093]

[0094] 3.2 First round of magnetic bead purification:

[0095] (1) Add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system after STEP3 is completed, mix by suction or vortex, and let stand at room temperature for 5 min.

[0096] (2) After the solution is clear after instant centrifugation, remove the supernatant, add 50 μL of YF buffer B to the tube, mix well, let stand at room temperature for 5 min, and then centrifuge briefly. Place the PCR tube on the DynaMag-96 Side magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, let stand for 30 s, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 s, discard the supernatant, centrifuge briefly, centrifuge the residual ethanol to the bottom of the tube, and discard the residual ethanol at the bottom.

[0097] (3) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and evaporate the residual ethanol. Add 24 μL of Nuclease-Free Water, pipette or vortex to mix, and let it stand at room temperature for 2 minutes.

[0098] (4) Centrifuge briefly until the solution is clear. Use a pipette to draw 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified multiplex PCR product. Label it and prepare for the second round of adapter sequence PCR reaction.

[0099] 3.3 Second round of adapter sequence PCR reaction

[0100] (1) The PCR reaction system includes the following reagents and amounts as shown in Table 6.

[0101] Table 6

[0102] system Volume (μL) Multiplex PCR amplification products 13.5 Enhancer buffer M 2.5 <![CDATA[ddH2O]]> 2 CDI primer (5μM) 2 IGT-EM808 polymerase mixture 10 Total volume 30

[0103] (2) Vortex the PCR tubes to mix them thoroughly, centrifuge briefly, and then place the PCR tubes into a thermal cycler and run the PCR program. The PCR program cycle parameters are shown in Table 7.

[0104] Table 7

[0105]

[0106] 3.4 Second round of magnetic bead purification

[0107] (1) Add 0.9 times the volume of pre-equilibrated and purified magnetic beads (27 μL) to the 30 μL reaction system after STEP3 is completed, mix by suction or vortex, and let stand at room temperature for 5 min.

[0108] (2) Centrifuge briefly, place the PCR tube on a magnetic rack and wait for the solution to clarify before completely removing the supernatant. Add 50 μL of YF buffer B to the tube, mix well, and let stand at room temperature for 5 min.

[0109] (3) Centrifuge briefly. Place the PCR tube on the DynaMag-96Side magnetic rack for 3 min, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, let stand for 30 s, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 s, discard the supernatant.

[0110] (4) Centrifuge briefly to remove the residual ethanol to the bottom of the tube and discard the residual ethanol at the bottom.

[0111] (5) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and evaporate the residual ethanol.

[0112] (6) Add 24 μL Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 min.

[0113] (7) Centrifuge briefly, place the PCR tube on a magnetic rack and wait for the solution to clarify. Take 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

[0114] 4. Quantitative Library

[0115] Take 1 μL of the prepared multiplex PCR library for use. Library concentration was determined and recorded using a 3.0 Fluorometer (Qubit dsDNA HSAssay Kit). The normal library concentration range is 5 ng / μL–20 ng / μL, and the concentration is primarily related to the template quality.

[0116] 5. Document Quality Inspection

[0117] The quality of all library fragments was assessed using the Agilent DNA 1000kit on an Agilent 2100 bioanalyzer. The library quality assessment results are shown below. Figure 1 As shown. By Figure 1 It can be seen that the target fragment distribution range of the library constructed by this invention is between 300bp and 450bp, which meets the relevant requirements.

[0118] 6. Illumina Miseq FGx TM Platform sequencing

[0119] Use Illumina Experiment Manager (IEM) software to set the sequencing parameters, including sample name, tag sequence, library preparation process, and sequencing cycle; add 600uL of 15.5pM library to the load sample well; and use RUO (research use only Run) mode for Miseq sequencing.

[0120] 7. Sequencing data processing

[0121] The raw sequencing data from the Miseq platform were analyzed using the STRait Razor v3 software, and then the data were summarized using R code to obtain the STR typing of the samples.

[0122] In the STait Razor v3 software, configuration files were set for each STR locus, including the flanking sequences at both ends of the locus's core sequence, the number of bases in the core repeat unit, and the number of bases in the core sequence that are not included in length polymorphisms. Based on these configuration file parameters, the STait Razor v3 software was used to perform preliminary analysis on each STR locus of the sample and output the results.

[0123] Based on the above output results, analyze the STR genotypes and then use R code to summarize the data. ① Set the analysis threshold to 5%, and mark low-coverage sequences below 5% as "Noise_S" (low coverage noise). ② In male samples, identify the most prevalent sequences as alleles. For female samples, calculate the coverage ratio of the two most prevalent sequences, and set the heterozygosity balance ratio (heterozygosity, Hb) threshold for STR loci: Hb ≥ 0.25 indicates heterozygosity, and Hb < 0.25 indicates homozygosity. Obtain the allele sequences for each STR locus of the sample. ③ For sequences with coverage exceeding the 5% analysis threshold but not identified as alleles, further determine whether they are stutter sequences based on sequence type. Sequences with one or fewer repeat units, or two or more repeat units than the allele sequences, are identified as "stutters," and the remaining sequences are identified as noise and marked as "Noise_L" (high coverage noise). Output the STR genotyping results for the sample.

[0124] The proportions of allele sequences, stutter sequences, "Noise_L" sequences, and "Noise_S" sequences in the total sequencing sequence (labeled as %Allele, %Stutter, %Noise_L, and %Noise_S, respectively) were calculated as indicators for sequencing performance evaluation.

[0125] II. Evaluate the sequencing results obtained from the above kits. The evaluation includes:

[0126] (1) Heterozygosity ratio of loci and sequence composition ratio

[0127] Table 8 shows the heterozygosity (Hb) and allele, stutter, "Noise_L", and "Noise_S" sequence composition of all STR loci.

[0128] Table 8

[0129]

[0130]

[0131] As shown in Table 8, DXS10147 had the highest heterozygosity ratio, while DXS6800 had the lowest. The average values ​​of %Allele, %Stutter, %Nois_L, and %Noise_S were 78.34%, 1.35%, 0.09%, and 20.21%, respectively.

[0132] (2) Consistency

[0133] Using Microreader TM The 19X ID System (Suzhou Yuewei Gene Technology Co., Ltd.) and the AGCU X PLUS kit (Wuxi Zhongde Meilian Biotechnology Co., Ltd.) were used to validate MPS typing of 30 loci of standard NA18530, namely DXS10074, DXS10075, DXS10079, DXS101, DXS10101, DXS10103, DXS10135, DXS10159, DXS10162, DXS6789, DXS6795, and DXS6. The following are listed: DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS981, DXS9895, DXS9902, GATA165B12, GATA172D05, GATA31E08, HPRTB, and DXS9907. Partial results of X-STR typing of the standard NA18530 are shown in Table 9.

[0134] Table 9

[0135]

[0136]

[0137] The results showed that the capillary electrophoresis (CE) typing of 30 X-STRs was completely consistent with the high-throughput sequencing (MPS) typing of this system. The remaining 22 loci were verified by Sanger sequencing, and the results were consistent with those of this system. This demonstrates that the sequencing results of the 52 STR loci in this detection system are accurate and reliable.

[0138] (3) Repeatability

[0139] Two libraries of DNA standard NA18530 were sequenced three times on the NovaSeq sequencing platform. After data analysis and comparison, the genotyping of the two libraries of NA18530 standard was consistent across 52 STR loci in the three sequencing runs. The sequencing depths were 60734×, 50964× and 51101×, respectively.

[0140] (4) Sensitivity

[0141] Based on the quantitative results of DNA standard NA18530, different template amount gradients (10ng, 5ng, 2ng, 1ng, 0.5ng, 0.25ng, 125pg, 61.5pg, 31.25pg, 15.625pg) were prepared and sequenced. Data analysis showed that when the DNA template amount was less than or equal to 125pg, the loci showed varying degrees of deletion but no incorrect typing. When the DNA template amount was greater than or equal to 0.25ng, the typing of each locus was complete and correct, indicating that the sensitivity of the typing system is not less than 0.25ng.

[0142] (5) Species specificity

[0143] Based on the Qubit quantification results of rabbit, mouse, sheep, pig and Escherichia coli samples, species-specific experiments were conducted. 0-2 loci were detected in each of the five samples, while all loci were genotyped in the standard NA18530 (human DNA), indicating that the system has good species specificity.

[0144] (6) Degraded samples

[0145] use The Pico ultrasonic disruptor (Diagenode, Belgium) was used to prepare DNA samples of different lengths for different ultrasonic times to simulate sample degradation. The ultrasonic frequency was set to 20-60 kHz, and the ultrasonic times were 0 min, 2.25 min, 3.75 min, 5 min, 10 min, 15 min, 20 min, and 30 min, respectively, to prepare eight degraded DNA samples ranging from 150 bp to 650 bp. Using the genotyping system provided by this invention, all loci were observed during ultrasonic disruption times of 0-15 min; one locus was missing during ultrasonic disruption times of 20-30 min.

[0146] (7) Inhibitor tolerance

[0147] Anti-inhibition experiments were conducted using different concentrations of humic acid (2 ng / μL, 5 ng / μL, 7 ng / μL, 10 ng / μL, 13 ng / μL) and hemoglobin (20 μM, 30 μM, 50 μM, 60 μM). DNA standard NA18530 was used for sequencing. Data analysis showed that the loci could be correctly detected when the humic acid concentration was less than or equal to 7 ng / μL and the hemoglobin concentration was less than or equal to 30 μM, indicating that the system has strong anti-inhibition ability.

[0148] (8) Mixture Analysis

[0149] Mixed male-to-male and female-to-male samples were constructed using DNA from NA18530 and 2800M, and F312 and NA18530 standards, respectively, and sequenced twice. The amount of DNA used was 20 ng. The detection rate of secondary contributor-specific alleles was observed at different mixing ratios (49:1, 39:1, 19:1, 9:1, 5:1, and 1:1). In the above mixtures, there were 46 and 34 minor contributor-specific alleles, respectively. The experimental results showed that when the male-to-male mixing ratio was less than or equal to 5:1, all of these minor contributor-specific alleles were detected. When the mixing ratio was greater than or equal to 9:1, the loss of minor contributor-specific alleles began to appear, with the number of lost sites ranging from 1 to 17. When the female-to-male mixing ratio was 1:1, all male-specific alleles, which are minor contributors, were detected. When the female-to-male mixing ratio was greater than or equal to 5:1, the loss of male-specific alleles, which are minor contributors, appeared, with the number of lost sites ranging from 1 to 16.

[0150] Example 2

[0151] This invention evaluates the identification efficacy of the X-STR typing systems provided in Examples 1 and 2, as detailed below:

[0152] Using the kit provided in Example 4, a population data survey was conducted on 128 unrelated healthy Han Chinese individuals from Hebei Province. There was no difference in X-STR frequencies between males and females. Hardy-Weinberg equilibrium test and linkage disequilibrium test were performed on the X-STR data of the female samples. 52 X chromosome STR loci were found to be in Hardy-Weinberg equilibrium and linkage equilibrium.

[0153] The forensic parameters of this detection system were calculated using two methods: (1) Based on the linkage disequilibrium test results, all 52 X-STRs provided in Example 1 of this invention were in linkage equilibrium and were calculated as 52 independent loci; (2) Following the 2017 ISFG guidelines on the use of X-STRs in kinship analysis (DNA Commission of the International Society for Forensic Genetics (ISFG) Guidelines on the use of X-STRs in kinship analysis), although linkage equilibrium between loci was demonstrated, due to sampling effects (such as small sample size), markers with similar genetic distances may erroneously reject linkage disequilibrium. Therefore, haplotype frequencies should be used. Considering the close proximity of the loci, this invention examined the efficacy of 47 X-STRs (i.e., the genotyping system containing 29 X-chromosome STR loci protected in Example 1) after excluding 5 loci. The 5 excluded loci were DXS680705, DXS680708, DXS680709, DXS10162, and DXS10163. Loci within 1 cM of each other were considered as a linkage cluster. The 47 X-STRs after excluding 5 loci comprised 29 X-chromosome STR loci, including 19 independent loci and 10 linkage clusters. See the description in Example 1 for details.

[0154] For the aforementioned 52 X-STRs, sequence polymorphism information was obtained based on their next-generation sequencing using the kit provided in Example 4. Capillary electrophoresis typing technology was used to obtain length polymorphism information, a commonly used method in the field of kinship identification. Therefore, based on the above sequence polymorphism information results and combined with the STR locus allele naming rules, this application statistically calculated the length polymorphism information of the corresponding X-STRs. The comparison of the number of alleles obtained based on length polymorphism and sequence polymorphism is shown in the figure below. Figure 2 As shown.

[0155] Depend on Figure 2 As can be seen, based on allele length polymorphism, a total of 416 alleles were obtained from the above 52 X-STRs; while based on allele sequence polymorphism, a total of 621 alleles were obtained from the above 52 X-STRs, with 23 loci showing an increase in alleles. Based on sequence polymorphism, DXS101 had the most alleles (63), while DXS7423 had the fewest (3). Taking the DXS10076 locus as an example, the alleles and frequencies detected based on length and sequence polymorphism are shown in Table 10. The allele frequencies in Table 10 are based on sequence polymorphism.

[0156] Table 10

[0157]

[0158]

[0159] As shown in Table 10, the kit containing 52 X-STR amplification primers provided in Example 4 of this invention can detect a greater number of alleles after sequencing. The typing system provided by this invention can improve the efficiency in kinship identification. For example, based on the length polymorphism allele 25, there are two sequence polymorphism alleles, namely [GAAG]10[GAAA]1[GAAG]4[GAAA]1[GAAG]4[AAAG]2[GAAG]3 and [GAAG]15[GAAA]1[GAAG]4[AAAG]2[GAAG]3. It can be seen that the kit provided in Example 4 of this invention can detect a greater number of alleles.

[0160] The haplotype frequencies of the above 10 linkage clusters were statistically analyzed based on 59 unrelated male individuals. The partial statistical table of haplotype frequencies of linkage cluster 5 based on sequence polymorphism is shown in Table 11.

[0161] Table 11

[0162]

[0163] Based on the allele frequencies and haplotype frequencies of sequence polymorphisms obtained from next-generation sequencing, the relevant forensic parameters of the X-STR loci of this invention were calculated, as shown in Tables 12-14. Table 12 shows the forensic parameters of 52 X-STR loci based on sequence polymorphisms, Table 13 shows the forensic parameters of 10 X-STR linkage clusters based on sequence polymorphisms, and Table 14 shows the system efficacy of different typing systems provided in Examples 1 and 2.

[0164] Table 12

[0165]

[0166]

[0167]

[0168] Note: Ho: Observed heterozygosity; He: Expected heterozygosity; DP_F: Female individual identification ability; DP_M: Male individual identification ability; TPE: Average exclusion probability of triplets; DPE_FF: Average exclusion probability of duos (mother and daughter); DPE_FM: Average exclusion probability of duos (mother and child, father and daughter).

[0169] Table 13

[0170]

[0171] Note: Ho: Observed heterozygosity; He: Expected heterozygosity; DP_F: Female individual identification ability; DP_M: Male individual identification ability; TPE: Average exclusion probability of triplets; DPE_FF: Average exclusion probability of duos (mother and daughter); DPE_FM: Average exclusion probability of duos (mother and child, father and daughter).

[0172] Table 14

[0173]

[0174]

[0175] Note: TDP f Women's cumulative individual identification ability; TDP m Cumulative individual identification ability for males; CTPE: Cumulative average exclusion probability of triplet; CDPE FF Cumulative average exclusion probability of twins (mother and daughter); CDPE FM Cumulative average exclusion probability of twins (mother and child, father and daughter)

[0176] As shown in Tables 12-14, the typing system containing 29 X chromosome STR loci provided by this invention can meet the system efficiency requirements for personal identification and kinship determination in forensic medicine, and provides a theoretical and experimental research basis for the application of the X chromosome STR system in forensic medicine to solve personal identification and kinship determination.

[0177] Example 3

[0178] The accuracy of the X-STR-containing typing systems provided in Comparative Example 1 and Examples 1-2 in second-degree kinship identification was compared using computer simulation. This verified the application value of the typing systems containing 29 X-chromosome STR loci provided in Examples 1 and 2 in complex kinship identification. Here, "complex kinship identification" mainly refers to the identification between kinship relationships at the same level that cannot be distinguished by autosomal genetic markers.

[0179] Taking the fine distinction of second-degree kinship as an example, the specific simulation process is as follows: (1) Draw a pedigree for each type of second-degree kinship and determine the marginal individuals, that is, samples whose alleles come entirely from unrelated individuals in the population, such as the grandmother and mother in the grandmother-granddaughter relationship; (2) Based on the allele / haplotype frequency data obtained in Effect Example 6, randomly generate the genotype of the marginal individuals; (3) According to the X chromosome inheritance law (that is, male individuals obtain one of their mother's two alleles with equal probability and pass it on to their daughters; female individuals obtain one allele from each of their parents and pass it on to their offspring with equal probability), generate the genotypes of other individuals. For example, in the grandmother-granddaughter relationship, one of the grandmother's two alleles is drawn with equal probability as the father's genotype, and at the same time as the granddaughter's paternal allele; one of the mother's two alleles is drawn with equal probability as the granddaughter's maternal allele; (4) Based on the genotype and allele / haplotype frequency data of the target individuals, calculate the specific likelihood ratio and conduct subsequent analysis to identify the kinship.

[0180] I. The follow-up analysis includes two methods: one is to predict the most likely group from multiple possible second-degree kinship groups. The analysis method is to calculate the likelihood ratio of each group relative to the relationship with unrelated individuals, and then perform a Bayesian transformation to obtain the post-probability. The formula for calculating the post-probability is shown in Equation 1.

[0181]

[0182] In the formula, the symbol "∝" indicates a direct proportional relationship; Pr(H x |E) represents the post-probability, that is, the probability of a specific hypothesis H given the detection of specific genetic evidence. x The probability that is true is Pr(H) x This is called the pre-probability, which is the probability of a specific hypothesis H without considering genetic evidence. x The probability that it is true; Pr(E|H) x This is called the likelihood, which is the determination of the hypothesis H. x Assuming the condition is true, the probability of detecting specific genetic evidence, after calculating the likelihood ratios of each group relative to unrelated individuals, is expressed as follows: this probability is related to the corresponding likelihood ratio LR. x Proportional.

[0183] If we calculate based on the assumption of equal probabilities, that is, assuming that all hypotheses are true with equal probability without considering genetic evidence, then we can deduce Equation 2.

[0184] Pr(H x |E)∝LR x Formula 2

[0185] The group corresponding to the calculated maximum likelihood ratio has the highest posterior probability. This is used as the criterion to classify cases into the corresponding kinship group.

[0186] Second, predict which of the two possible kinship groups (A and B) is more likely. The analysis method is to calculate the likelihood ratio of group A to group B, and use a likelihood ratio of 1 as the discrimination threshold. If the likelihood ratio is greater than 1, it is classified as group A, otherwise it is classified as group B.

[0187] Results of grouping and screening analysis of second-degree kinship.

[0188] (2-1) In this invention, second-degree kinship is divided into three categories: female-female, male-female, and male-male. Each category is further divided into different groups. Based on the X chromosome locus, the above three categories of second-degree kinship are further divided into 10 groups (containing 22 types) of kinship, as follows:

[0189] (2-1-1) Fine distinction of female-female second-degree kinship

[0190] Female-female second-degree kinship is divided into three groups: Group 1: grandmother-granddaughter, half-sisters; Group 2: aunt-niece; Group 3: maternal grandmother-granddaughter, half-sisters, aunt-niece. These relationships are difficult to distinguish using autosomal loci.

[0191] (2-1-2) Fine distinction of male-female second-degree kinship

[0192] Second-degree male-female kinship is divided into the following four groups: Group 4: Aunt-nephew; Group 5: Half-siblings (same mother, different fathers), maternal grandmother-grandson, maternal grandfather-granddaughter, uncle-niece; Group 6: Uncle-niece; Group 7: Grandmother-grandson, grandfather-granddaughter, aunt-nephew, half-siblings (same father, different mothers). It is difficult to determine the specific kinship category of these relationships using autosomal loci.

[0193] (2-1-3) Fine distinction of male-male second-degree kinship

[0194] Male-male second-degree kinship is divided into the following three groups: Group 8: half-brothers (same mother, different father), maternal grandfather-grandson; Group 9: maternal uncle-nephew; Group 10: paternal grandfather-grandson, maternal uncle-nephew, half-brothers (same father, different mother). It is difficult to determine the specific kinship category of these relationships using autosomal loci.

[0195] (2-2) Simulate 1 million pairs of each of the above-mentioned kinship relationships. For example, Group 1 includes two kinship relationships: grandmother-granddaughter and half-sisters. A total of 2 million pairs are simulated. For each simulated pair, calculate three or four cumulative likelihood ratios corresponding to three or four groups. The group with the highest value is used as the predicted value. The accuracy of the output results is used to evaluate the application value of the typing system containing 29 X chromosome STR loci provided in Embodiment 1 or 2 of this invention in the identification of secondary kinship.

[0196] Specifically, the kits provided in Examples 3 or 4 were used to analyze the genetic information at the corresponding loci. The confusion matrix diagram for finely distinguishing 22 second-degree relationships using the genotyping system containing 29 X-chromosome STR loci provided in Example 1 is shown below. Figure 3 As shown, the confusion matrix diagram for finely distinguishing 22 secondary kinships using the genotyping system containing 29 X chromosome STR loci provided in Example 2 is shown below. Figure 4 As shown.

[0197] (2-2-1) For female-female second-degree kinship, calculate the three cumulative likelihood ratios for each simulated sample pair, corresponding to three groups, and output the predicted value of the group with the maximum value.

[0198] Depend on Figure 3-4 It can be seen that, using the typing system of Embodiments 1-2 of the present invention, all 2 million pairs of the two relationship samples in Group 1 were correctly identified. Regarding Group 2 and Group 3, the typing system containing 47 X-STRs provided in Embodiment 1 was correctly identified in 81.58% and 82.03% of cases, respectively, while the typing system containing 52 X-STRs provided in Embodiment 2 was correctly identified in 86.20% and 85.35% of cases, respectively. Therefore, it is evident that the typing systems provided in Embodiments 1-2 of the present invention can effectively distinguish individual pairs within the three groups of female-female second-degree kinship.

[0199] (2-2-2) Regarding male-female second-degree kinship, the genetic information of 1 million pairs of each of the above four kinship groups at the loci provided in the kits of Example 3 or 4 was simulated. Using the allele frequency data of the genotyping system provided in Example 1 or 2, four groups were calculated for each simulated individual pair, corresponding to four likelihood ratios (where the likelihood ratio corresponding to Group 7 was counted as 1), and the group corresponding to the maximum value was output as the predicted value.

[0200] Depend on Figure 3-4It can be seen that, using the genotyping system containing 47 X-STRs provided in Application Example 1, Groups 4-7 were correctly identified at rates of 86.58%, 69.40%, 73.34%, and 91.79%, respectively. Using the genotyping system containing 52 X-STRs provided in Application Example 2, Groups 4-7 were correctly identified at rates of 91.17%, 78.68%, 79.08%, and 92.43%, respectively. Therefore, it is evident that the genotyping systems provided in Examples 1-2 of this invention can effectively distinguish between individual pairs in the four groups within a male-female second-degree kinship relationship.

[0201] (2-2-3) For male-male second-degree kinship, the genetic information of 1 million pairs of each of the above three kinship relationships at the kit loci provided in Example 3 or 4, and the allele frequency data of the typing system provided in Example 1 or 2 are simulated respectively. For each simulated individual pair, three groups are calculated corresponding to the three likelihood ratios (where the likelihood ratio corresponding to Group 10 is counted as 1), and the group corresponding to the maximum value is output as the predicted value.

[0202] Depend on Figure 3-4 It can be seen that, using the genotyping system containing 47 X-STRs provided in Example 1, Groups 8-10 were correctly identified at 86.63%, 79.63%, and 94.85% respectively; using the genotyping system containing 52 X-STRs provided in Example 2, Groups 8-10 were correctly identified at 91.93%, 87.03%, and 96.10% respectively. Therefore, it is evident that the genotyping systems provided in Examples 1-2 of this invention can effectively distinguish individual pairs within the three groups of male-male second-degree kinship.

[0203] The analysis results of the two relationships show that, considering the gender combination between individuals, there are 12 kinds of one-to-one identification needs among the 10 groups. If the likelihood rate is equal to 1 as the judgment threshold, the accuracy, sensitivity, specificity, positive prediction probability and negative prediction probability of each group of kinship identification are shown in Table 15.

[0204] Table 15

[0205]

[0206]

[0207] Note: SEN: Sensitivity (probability of correctly predicting group A), SPE: Specificity (probability of correctly predicting group B), PPV: Positive Prediction Probability (probability of being predicted as A), NPV: Negative Prediction Probability (probability of being predicted as B), ACC: Accuracy (probability of correctly predicting A and B).

[0208] (2-2-4) Using the genotyping system containing 32 X-STRs provided in Comparative Example 1, simulations were performed according to the above method, and a discrimination analysis of second-degree kinship was conducted. The confusion matrix diagram for finely distinguishing 22 types of second-degree kinship using the genotyping system containing 32 X-STRs provided in Comparative Example 1 is shown below. Figure 5 As shown.

[0209] Depend on Figure 5 As can be seen, compared with the classification system containing 52 X-STRs provided in Example 2, the classification system containing 32 X-STRs provided in Comparative Example 1 has the same identification accuracy for Group 1, while the identification accuracy for Groups 2-10 is 75.69%, 78.45%, 85.12%, 65.20%, 65.69%, 87.36%, 85.46%, 74.76%, and 91.74%, respectively. The classification system provided in Example 2 of this invention has a significantly higher average identification accuracy (88.9%) for the 10 groups than Comparative Example 1 (81.5%).

[0210] In summary, the X-STR typing system provided in Embodiment 1 or 2 of this invention has higher accuracy compared to other X-STR typing systems. It can play an auxiliary role in the identification of complex kinship, especially in the further fine differentiation of secondary kinship, where it can play an irreplaceable role compared to autosomal loci.

[0211] Typical Case 1

[0212] This invention applies the 29-group X-STR typing system to a case of grandmother-granddaughter identification. Due to an inheritance dispute, it was necessary to identify two female individuals to determine if one was the biological grandmother of the other. A total of 42 autosomal STR loci from the three STR complex systems were examined (D6S477, D18S535, D19S253, D15S659, D11S2368, D20S470, D1S1656, D22-GATA198B05, D16S539, D7S3048, D8S1132, D4S2366, D21S1270, D13S325, D9S925, D3S3045, D14S608, D10S1435, D12S391, D2S1338, D17S1290, D5S...). After considering the genotype distribution frequencies of the above autosomal STR loci in the Han Chinese population of Hebei Province and the genotypes of the two individuals, the grandparent-grandchild index between the two individuals was calculated using the ITO method to be 7.9906. After examining the two individuals using the genotyping system containing 29 X-STRs provided in Example 1 of this invention, the haplotypes of the two individuals on the 10 X-STR linkage clusters provided in Example 1 were estimated using FamlinkX software. Based on the allele frequency data distribution of these 29 X-STRs, the likelihood ratio corresponding to the grandmother-granddaughter relationship between the two individuals was calculated to be 1.66 × 10⁻⁶. 10 The results are significantly greater than those obtained from the aforementioned 42 autosomal STRs. Therefore, the X-STR testing system provided by this invention can provide more reliable evidence in identifying grandmother-granddaughter relationships compared to autosomal STR systems of similar magnitude.

[0213] Typical Case 2 An example of using the 29-group X-STR typing system provided by this invention for identifying kinship at the same level. During the investigation of a criminal case, a sample was found at the scene. After investigation, it was determined that the person who left the sample might be one of a father and son (father A or son B). It was necessary to determine whether the sample was left by individual A or individual B. Further investigation revealed that the person who left the sample was a second-degree kinship member. Figure 6As shown, only one female individual, C (granddaughter of individual A and niece of individual B), was available for comparison in the family pedigree. Analysis revealed that the kinship between individual C and both individuals A and B was two-tiered, indistinguishable using independently inherited autosomal genetic markers. However, from an X-chromosome inheritance perspective, the relationship between individual C and individual A belongs to Group 7 as described in this invention, while the relationship between individual C and individual B belongs to Group 5. After examining the two individuals using the genotyping system containing 29 X-STRs provided in Example 1 of this invention, the haplotypes of individual C and the individuals from which the field-exposed sample originated were estimated using FamlinkX software on 10 X-STR linkage clusters within the 29 X-STRs. Based on the allele frequency data distribution of these 29 X-STRs, the likelihood ratio of Group 5 relative to Group 7 was calculated to be 3.18 × 10⁻⁶. 4 They tend to believe that the person remaining in the sample is individual B.

[0215] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A typing system comprising 29 X-chromosome STR loci, characterized in that, The 29 sets of X chromosome STR loci include 19 independent X-STRs and 10 X-STR linked clusters; Among them, 19 independent X-STRs include DXS9902, DXS6795, DXS9907, DXS6810, GATA144D04, DXS981, DXS6800, DXS6803, DXS9898, DXS9905, DXS6801, DXS6799, DXS7133, DXS6804, GATA172D05, DXS7130, GATA165B12, GATA31E08 and DXS7127; The 10 X-STR linkage clusters include 10 linkage clusters, from linkage cluster 1 to linkage cluster 10. Cluster 1 includes DXS680703, DXS6807, DXS680706, and DXS680710; Cluster 2 includes DXS9895 and DXS9906; Cluster 3 includes DXS10135 and DXS8378; Cluster 4 includes DXS10076 and DXS10078; Cluster 5 includes DXS10161, DXS10160, and DXS10159; Cluster 6... The chain includes DXS7132, DXS10079, DXS10074 and DXS10075, chain 7 includes DXS6809 and DXS6789, chain 8 includes DXS7424 and DXS101, chain 9 includes DXS10103, HPRTB and DXS10101, and chain 10 includes DXS8377, DXS10146, DXS10147 and DXS7423.

2. The typing system comprising 29 X chromosome STR loci as described in claim 1, characterized in that, The typing system also includes five X chromosome STR loci, specifically DXS10162, DXS10163, DXS680705, DXS680708, and DXS680709.

3. A primer composition for a typing system comprising 29 X chromosome STR loci as described in claim 1 or 2, characterized in that, The primer composition includes primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

94.

4. The primer composition of the typing system containing 29 X chromosome STR loci as described in claim 3, characterized in that, The primer composition also includes primers with nucleotide sequences as shown in SEQ ID NO.95 to SEQ ID NO.

104.

5. A reagent kit, characterized in that, Includes primer compositions for amplifying the genotyping system comprising 29 X chromosome STR loci as described in claim 1 or 2.

6. The reagent kit as described in claim 5, characterized in that, The primer composition includes primers shown in SEQ ID NO.1 to SEQ ID NO.94 or primers shown in SEQ ID NO.1 to SEQ ID NO.

104.

7. The application of the typing system containing 29 X chromosome STR loci as described in claim 1 or 2 in non-diagnostic individual identification or kinship identification.

8. The application as described in claim 7, characterized in that, The kinship relationship described is a second-degree kinship.

9. A method for identifying kinship for non-diagnostic purposes, characterized in that, The identification method includes the following steps: DNA was extracted from two samples to be tested, and the genotyping system containing 29 X chromosome STR loci as described in claim 1 or 2 was amplified. After sequencing on a next-generation sequencing platform, the data was analyzed to obtain the X chromosome STR locus allele genotypes of the samples and calculate the likelihood rate. The kinship was identified based on the likelihood rate results.

10. The method for identifying kinship for non-diagnostic purposes as described in claim 9, characterized in that, The extraction of DNA from the two test samples, amplification of the genotyping system containing 29 X chromosome STR loci as described in claim 1 or 2, and analysis of the data and calculation of likelihood after sequencing on a next-generation sequencing platform specifically include: extracting and quantifying DNA from the peripheral blood of the two test samples; using the kit described in claim 5 or 6, performing a first round of multiplex PCR amplification and a second round of adapter sequence PCR reaction on the DNA of the test samples; subsequently quantifying and quality-testing the library; sequencing on a next-generation sequencing platform; analyzing and obtaining the X chromosome STR locus allele genotyping of the samples; and calculating the likelihood based on the population frequency of the corresponding alleles.

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